Muscle contraction requires the orderly assembly and functional interaction of numerous proteins, many of which are represented by multiple isoforms. The focus of our proposal is the motor molecule, myosin, which is encoded by at least 10 genes in mammals. The extent of the myosin gene family is still not known; nor is it clear what impact myosin composition has on the development and function of muscle. We propose to use yeast artificial chromosomes to define the human skeletal muscle myosin heavy chain (MHC) locus on chromosome 17. We will determine the order, orientation and identity of the 10 MHC genes we have found. One of myosin's hallmark functions is its self-assembly into thick filaments. The sequences responsible for myosin self assembly are beginning to be understood, but it is clear that reconstituted myosin filaments differ in length and composition from native thick filaments. We will investigate the determinants of myosin thick filament assembly and the roles of thick filament-associated proteins in this process, using a cell culture transfection system. in addition, we will characterize the impact of myosin mutations on thick filament formation and function in the context of both isolated cells and the intact muscle. An important question is whether myosin gene diversity provides functional specialization, or whether it represents redundancy within the system. To address this question, we will use homologous recombination in embryonic stem cells to inactivate the perinatal skeletal MHC gene in mice. This will allow us to determine whether the perinatal MHC gene is essential for normal muscle development and function. The identification of myosin mutations in the autosomal dominant heart disease, familial hypertrophic cardiomyopathy (FHC) has led us to use transgenic approaches to ask the following 2 questions: 1. Will mutations in skeletal myosin that are analogous to those found in FHC result in skeletal muscle disease? 2. Will mutation of known functional domains in the cardiac MHC molecule result in a dominant phenotype similar to that seen in FHC? These approaches should allow us to understand a number of important aspects of muscle development, structure and function.